Whole fetal bovine ovaries were homogenised in 1 ml Trizol® (Thermo Fisher Scientific, Waltham, MA, United States) using the Mo Bio Powerlyser 24 (Mo Bio Laboratories Inc., Carlsbad, CA, United States) and RNA extracted according to manufacturer’s instructions. All samples were treated with DNase I (Promega/Thermo Fisher Scientific Australia Pty Ltd., Tullmarine, Vic, Australia). The RNA concentration and quality (RQI, Table 2 ) was then determined using the Experion™ RNA StdSens Analysis kit and the Experion™ Automated Electrophoresis System (Bio-Rad Laboratories Pty., Ltd., Gladesville, NSW, Australia). 500 ng/50 µl per well (96-well plate) of total RNA of each sample was used for RNA-seq.
RNA-seq based transcriptome profiling was performed at the SAHMRI Genomics Facility (SAHMRI, Adelaide, SA, Australia). Briefly, Single-end Poly A-selection mRNA libraries (∼35 M reads per sample) were created using the Nugen Universal Plus mRNA-Seq library kit from Tecan (Mannedorf, Switzerland) and sequenced with an Illumina Nextseq 500 using single read 75 bp (v2.0) sequencing chemistry (Illumina Inc., San Diego, CA, United States). Two sequencing runs, with 10 samples per run, were performed and sample 15/R43t was used as internal control in both runs.
RNA-seq based transcriptome profiling was performed at the SAHMRI Genomics Facility (SAHMRI, Adelaide, SA, Australia). Briefly, Single-end Poly A-selection mRNA libraries (∼35 M reads per sample) were created using the Nugen Universal Plus mRNA-Seq library kit from Tecan (Mannedorf, Switzerland) and sequenced with an Illumina Nextseq 500 using single read 75 bp (v2.0) sequencing chemistry (Illumina Inc., San Diego, CA, United States). Two sequencing runs, with 10 samples per run, were performed and sample 15/R43t was used as internal control in both runs.